Truncation of CD148 and CD45 ectodomains enhances colocalization with TCR microclusters. (A) B3Z T cells expressing the indicated CD148-GFP chimera (green) were imaged by TIRF microscopy after initial contact with a planar bilayer that contained glycosylphosphatidylinositol-ICAM-1 and Cy5-labeled (red) monobiotinylated anti-mouse CD3ε. Representative images are shown (upper). Pearson correlation coefficients between the GFP and Cy5 fluorescence were analyzed in 20 cell contacts (lower). (B) B3Z T cells expressing CD2-TCRζ-mCherry and the indicated CD148-GFP chimera were preincubated with or without PP2 and then imaged in contact with a planar bilayer that contained glycosylphosphatidylinositol-ICAM-1 and anti-rat CD2. Pearson’s correlation coefficients between GFP and mCherry fluorescence were analyzed in 20 cell contacts. (C) 2B4 cells expressing the indicated CD45-GFP chimera (green) were preincubated with or without PP2 and imaged by TIRF microscopy after initial contact with a planar bilayer that contained ICAM-1 and monobiotinylated anti-mouse CD3ε. CD45 protein was imaged through its C-terminal GFP tag, and TCRβ was detected using a specific antibody (H57) Fab′ fragment labeled with AF568 (upper). Pearson’s correlation coefficients between the GFP and AF568 were analyzed on more than 25 cell contacts (lower). ***P < .01, using Student t test.